Over the last several decades, mass spectrometry has emerged as one of the most broadly applicable analytical tools for detection and characterization of a wide class of molecules. Mass spectrometric analysis is applicable to almost any chemical species capable of forming an ion in the gas phase, and, therefore, provides perhaps the most universally applicable method of quantitative analysis. In addition, mass spectrometry is a highly selective technique especially well suited for the analysis of complex mixtures comprising a large number of different compounds in widely varying concentrations. Further, mass spectrometric analysis methods provide high detection sensitivities, approaching tenths of parts per trillion for some species. As a result of these beneficial attributes, a great deal of attention has been directed over the last several decades at developing mass spectrometric methods for analyzing complex mixtures of biomolecules, such as peptides, proteins and oligonuceotides and complexes thereof.
Mass spectrometric analysis involves three fundamental processes: (1) gas phase ion formation, (2) mass analysis whereby ions are separated on the basis of mass-to-charge ratio (m/z), and (3) detection of ions subsequent to separation. The overall efficiency of a mass spectrometer (overall efficiency=(analyte ions detected)/(analyte molecules consumed)) may be defined in terms of the efficiencies of each of these fundamental processes by the equation:EMS=EF×EMA×ED,  (I)where EMS is the overall efficiency, EF is the ion formation efficiency (ion formation efficiency=(analyte ions formed)/(analyte molecules consumed during ion formation)), EMA is the mass analysis efficiency (mass analysis efficiency=(analyte ions mass analyzed)/(analyte ions consumed during analysis)) and ED is the detection efficiency (detection efficiency=(analyte ions detected)/(analyte ions consumed during detection)).
Despite the fact that mass spectrometry is currently one of the most widely used techniques for identifying and characterizing biomolecules, conventional state of the art mass spectrometers have surprisingly low overall efficiencies for these compounds. For example, a quantitative evaluation of the efficiency of a conventional orthogonal injection time-of-flight mass spectrometer (Perseptive Biosystems Mariner) for the analysis of a sample containing a 10 kDa protein yields the following efficiencies, ES=1×10−4, EMA=8×10−7, and ED=9×10−3, providing an  overall efficiency of the mass spectrometer of 1 part in 1012. As a result of low overall efficiency, conventional mass spectrometric analysis of biomolecules typically requires large samples and is unable to achieve the ultra low sensitivity needed for many important biological applications, such as single cell analysis of protein expression and post-translational modification. Therefore, there is a significant need in the art for more efficient ion preparation, analysis and detection techniques to capture the full benefit of mass spectrometric analysis for important biological applications.
Over the last decade, new ion preparation methods have revolutionized mass spectrometric analysis of biological molecules. These new ionization methods, which include matrix assisted laser desorption and ionization (MALDI) and electrospray ionization (ESI), provide greatly improved ionization efficiency for a wide range of compounds having molecular weights up to several hundred kiloDaltons. Moreover, MALDI and ESI ionization sources have been successfully integrated with a wide range of mass analyzers, including quadrupole mass analyzers, time-of-flight instrumentation, magnetic sector analyzers, Fourier transform—ion cyclotron resonance instruments and ion traps, to provide selective identification of polypeptides and oligonucleotides in complex mixtures. Mass determination by time-of-flight (TOF) analysis has proven especially well suited for the high molecular weight biomolecules ionized by ESI and MALDI techniques because TOF has no intrinsic limit to the mass range accessible, provides high spectral resolution and has fast temporal response times. Use of time-of-flight mass analysis with ESI and MALDI ion sources for proteomic analysis is described in detail by Yates in Mass Spectrometry and the Age of the Proteome, Journal of Mass Spectrometry, Vol. 33, 1-19 (1998). As a result of these advances, MALDI-TOF and ESI-TOF have emerged as the two most commonly used ionization techniques for analyzing complex mixtures of biomolecules having high molecular weights.
Although integration of modern ionization techniques and time-of-flight analysis methods has greatly expanded the mass range accessible by mass spectrometric methods, complementary ion detection methods suitable for time of flight analysis of high molecular weight compounds, including many biological molecules, remain considerably less well developed. Indeed, the effective upper limit of mass ranges currently accessible by MALDI-TOF and ESI-TOF analysis techniques are limited by the sensitivity of conventional ion detectors for high molecular weight ions. For example, conventional multichannel plate (MCP) detectors exhibit detection sensitivities that decrease significantly with ion velocity, which corresponds to a decrease in sensitivity with increasing molecular weight when these detectors are used for TOF mass analysis.
MCP detectors are perhaps the most pervasive ion detector used in ESI-TOF and MALDI-TOF mass spectrometry. These detectors operate by secondary electron emission and typically comprise a parallel array of miniature channel electron multipliers. Typically the channel diameters are in the range of 10 to 100 microns with the lengths of the channels in the neighborhood of 1 mm. Each channel operates as a continuous dynode structure, meaning that it acts as its own dynode resistor chain. A potential of about 1 to 2 kV is placed across each channel. When an energetic molecule enters the low potential end of the channel and strikes the wall of the channel it produces secondary electrons which are in turn accelerated along the tube by the electric field. These electrons then strike the wall generating more electrons. The process repeats many times until the secondary electrons emerge from the high potential end of the channel. Generally speaking for each molecule which initiates a cascade, 104 electrons emerge from the channel providing significant gain. The electron cascade formed is collected at an anode and generates an output signal. MCP detectors can be made in large area format which is useful for analysis of packets of ions in TOF systems.
A number of substantial limitations of this detection technique arise out of the impact-induced mechanism of MCP detectors governing secondary electron generation. First, the yield of secondary electrons in a MCP detector decreases significantly as the velocities of ions colliding with the surface decreases. As time-of-flight detectors accelerate all ions to a fixed kinetic energy, high molecular weight ions have lower velocities and, hence, lower probabilities of being detected by MCP detectors. Second, the secondary electron yield of MCP detectors also depends on the composition and structure of colliding gas phase ions. Third, it is also well established that once a cascade has been initiated within a channel, it is depleted of electrons. Due to the high resistivity of the channel, the time required to replace these electrons is several orders of magnitude larger (milliseconds) than the duration of the TOF measurement (microseconds). Thus, for a single TOF event a channel is rendered inactive after a single cascading event, thus each successive packet of ions impinging on the detector has fewer and fewer active channels available to it.
As is apparent to those skilled in the art of mass spectrometry, the limitations associated with MCP detectors restrict the mass range currently accessible by MALDI-TOF and ESI TOF techniques, and hinder the quantitative analysis of samples comprising complex mixtures of high molecular weight biopolymers. Accordingly, there currently exists a need for ion detectors that do not exhibit decreasing sensitivities with increasing molecular weight and that do not have sensitivities dependent on the composition and structure of gas phase ions analyzed.
Over the last decade, considerable research has been directed at developing new sensors based on nanoelectromechanical resonators that are suitable for detecting and analyzing high molecular weight compounds. U.S. Pat. No. 6,722,200, for example, describes mass analyzers for use in mass spectrometry analysis comprising an array of nanoelectromechanical beam resonators. In these systems, the fundamental mode resonance frequencies of a plurality of double-clamped nanoelectromechanical beam resonators are monitored in time using a phase-locked loop circuit. The surfaces of the beam resonators are positioned to intersect the path of a stream of gas phase molecules to be detected. Collisions between the molecules and the surface of a beam resonator results in accommodation which in turn provides a measurable change of the resonance frequency of the resonator. The measured change in resonance frequency is reported to be related to the mass of the molecule(s) received by the resonator and, hence continuously monitoring the resonance frequencies of resonators in the array provides a means of detecting and analyzing molecules.
Although the sensor designs disclosed in U.S. Pat. No. 6,722,200 are reported to provide a sensitive means of detecting and analyzing molecules, particularly high molecular weight molecules, this technique is susceptible to a number of drawbacks that make its integration with conventional mass spectrometry systems impractical. First, 10s of thousands of resonators are needed to provide a detector with a large enough active area for use in a conventional TOF mass spectrometer. Individually reading out each resonator in such a large array is expected to take a very long time and thus, these sensors are not likely to provide a temporal response time useful for most mass spectrometry systems, such as TOF analyzers. Further, such a readout system is expected to be cumbersome (difficult to house in a compact fashion) and cost prohibitive for commercial development. Second, the measured change in resonance frequency is expected to depend significantly on the exact location on the resonator where contact is established with the molecule(s) undergoing detection/analysis. This dependency is likely to result in significant variations in detection sensitivity and mass resolution as a function of where the molecule contacts the resonator. Finally, removal of the molecule after detection to ready the device for another detection event requires post detection processing, such as elevating the temperature, providing electromagnetic radiation and/or treatment by other thermal means. These processes are expected to materially change the physical dimensions and composition of resonators in the array, particularly given their incredible small physical dimensions. Therefore, these post detection processes are likely to undermine the performance reliability of these devices with respect to sensitivity and resolution.
It will be appreciated from the foregoing that there is currently a need in the art for methods, systems and devices for detecting and analyzing molecules having large molecular masses. Specifically, detection methods and systems providing sensitive detection of large molecular mass molecules are needed that are capable of effective integration with conventional mass spectrometry systems, such as TOF analysis systems. Sensors and analyzers are needed for mass spectrometry applications that do not exhibit deceases in sensitivity as a function of molecular mass, and that are capable of fast readout and good temporal resolution.